Maintenance of Hybridomas in Rodents and the Collection of Ascites Fluid

Policy

The Institutional Animal Care and Use Committee (IACUC) and Animal Care Program (ACP) have developed the following policy intended to eliminate or reduce to a minimum animal discomfort associated with in vivo production of monoclonal antibodies. When proposing to use the rodent ascites method of monoclonal antibody production, investigators must include the following information in the animal care and use protocol:

• Confirmation that the procedures to be used may cause discomfort, distress, or pain.
• Description of the search for alternative methods.
• Scientific justification of the need for using the in vivo method including an explanation of why alternative in vitro methods cannot be used.

Protocols should explicitly describe hybridoma procedures, monitoring, humane endpoints, and training of research personnel.

Background

Monoclonal antibodies represent a powerful research tool, and the use of animals is indispensable to the establishment of monoclonal antibody-producing cell lines. Once these cell lines are established, in vivo and/or in vitro techniques can be utilized for the production of the necessary quantity of monoclonal antibodies. There is evidence that the rodent ascites method of monoclonal antibody production causes discomfort, distress, and/or pain, and practical in vitro methods exist that can replace the ascites method in many experimental applications. In vitro methods should be used whenever feasible. There is, however, scientific justification for using in vivo methods for producing monoclonal antibodies in certain situations.

Procedures for Ascites Production

Priming

The biological effects of the commonly used priming agent, pristane include granulomatous inflammation, lymphatic obstruction, and reduced clearance of cells from the peritoneal cavity. The volume of pristane injected intraperitoneally (IP) should be reduced to the smallest volume necessary to stimulate the formation of ascites fluid. Lower doses of pristane have been associated with fewer adverse clinical signs and improved animal welfare.

• An IP injection volume of 0.1-0.3 mL or pristane should be used.
• Scientific justification must be provided to use doses larger than 0.3 mL.
• Incomplete Freund’s Adjuvant (IFA) may also be used for priming at a dose of 0.25-0.50 mL IP

Hybridoma Testing

Externally generated immortal cell lines and rodent-derived biologics that are to be used in hybridoma formation should be tested for the presence of rodent viruses prior to introduction into the animal colony. Please contact the Animal Care Program Policy Testing Biological Specimens to be Implanted into Live Laboratory Rodents.

Hybridoma Implantation

• The recommended time interval between pristine priming and inoculation of hybridoma cells ranges from three days to three to four weeks.
• Hybridoma cells should be injected at a volume of <0.5 mls IP.
• The total number of cells in the inoculum should range between 105-107 cells.
• A baseline body weight must be taken at the time of tumor cell injection

Post-Hybridoma Implantation Monitoring

• After inoculation with an ascites-producing tumor line, research staff should observe rodents at least every other day for the first five days, then daily thereafter.
• Research staff must be trained appropriately to monitor for clinical signs associated with ascites production (e.g. abdominal distention, respiratory difficulty), as well as signs of distress (e.g. ruffled fur, inappetence, poor body condition, constipation, diarrhea, impaired ambulation). 
• Mice must be weighed on the day of priming, and then daily beginning by day 5. Ascites fluid must be removed before a 20% weight gain is noted.
• Veterinary staff must be notified of any signs of pain and distress are identified.

Collect of Ascites Fluid

• Fluid should be collected before abdominal distention becomes severe enough to cause discomfort or impair normal activity or before 20% weight gain is obtained.
• Ascites fluid collection by abdominal paracentesis should be performed by trained personnel. ACP staff are available to provide training.
• Manual restraint or anesthesia may be utilized for collection procedures. Anesthesia should be used when training individuals in this technique.
• Aseptic technique is recommended for the preparation of the collection site.
• An 18-22-gauge needle should be used.
• Fluid should be removed slowly in order to minimize the possibility of shock.
• Animals must be monitored continuously for 20-30 mins after ascites collection for signs of shock (e.g. pale eyes, ears, and muzzle, weakness).
• Warmed saline or lactated ringers may be given to prevent or treat shock. Two to three mls of warmed fluid should be given subcutaneously immediately prior to collection or when treating shock. The animal should then be monitored for an additional 20-30 mins.

Ascites Collection Frequency

• The time intervals between ascites collection should be as short as possible. One to three days is the recommended frequency.
• A total of three abdominal paracenteses may be performed. A fourth paracentesis may be performed after the animal is euthanized.

Human Endpoints

Animals must be euthanized if any of the following criteria are met:

• Abdominal paracentesis does not relieve abdominal distension or respiratory difficulty.
• Animal exhibits one or more of the following criteria: hunched posture, lethargy, inability to rise or ambulate, respiratory difficulty.
• Weight of animal is >25% over baseline due to ascites volume.
• Animals have hemorrhagic and/or cloudy ascites fluid.

Responsibilities

Role of the Principal Investigator (PI): The investigator is responsible for justifying the appropriate methods to be used on a project-by-project basis and approved prior to initiation of project work. Investigators are strongly encouraged to consult with veterinary staff during protocol development. The PI is also responsible for training project personnel in all aspects of hybridoma ascites production and animal monitoring listed above.

Role of ACP: The veterinary staff must approve the proposed methods and monitoring procedures to minimize pain and distress associated with hybridomas and ascites production. ACP staff may also provide training in handling, injection, abdominal paracentesis, and clinical monitoring.

Role of the IACUC: Federal regulations require the IACUC to critically evaluate the proposed use of the mouse ascites method of monoclonal antibody production on a project-by-project basis. Prior to approval of proposals, which include this method, the IACUC must determine that (i) the proposed use is scientifically justified, (ii) methods that avoid or minimize discomfort, distress, and pain (including in vitro methods) have been considered, and (iii) the latter have been found unsuitable for protocol-specific scientifically-based reasons. The IACUC fulfills this evaluation during the critical review of newly submitted animal care and use protocols and of amendments to previously approved protocols.

References

• Brodeur, B.R., Tsang, P. and Larose, Y. Parameters affecting ascites tumor formation in mice and monoclonal antibody production. Journal of Immunological Methods 71:265-272, 1984.
• Canadian Council on Animal Care. Guidelines on: Antibody Production, 2002. 
• ILAR, Monoclonal Antibody Production, A Report of the Committee on Methods of Producing Monoclonal Antibodies, National Research Council, 1999.
• Jackson LR, Fox JG. Institutional policies and guidelines on adjuvants and antibody production. ILAR Journal 37(3): 141-152, 1995.
• John Hopkins Center for Alternatives to Animal Testing. 
• Lipman N.S., L.R. Jackson, L.J. Trudel, and F. Weis-Garcia. Monoclonal versus polyclonal antibodies: Distinguishing characteristics, applications, and information resources. ILAR Journal 46(3): 258-268, 2005. 
• Moore,J.M., and T.V. Rajan. Pristane retards clearance of particulate materials from the peritoneal cavity of laboratory mice. J. Immunological Methods 173:273-278, 1994.
• Peterson N.C. Behavioral, clinical and physiologic analysis of mice used for ascites monoclonal antibody production. Comparative Medicine 50(5): 516-526, 2000.
• Workman P. et al. Guidelines for the welfare and use of animals in cancer research. British Journal of Cancer 102: 1555-1577, 2010.